Vaccines containing various living avirulent cells of tubercle bacilli, in particular Bacillus Calmette-Guerin (BCG), an attenuated bovine tuberculosis organism (Mycobacterium bovis), have been used to induce immunity against acquired tuberculosis disease in man. Recently, BCG vaccine has been used as an immunostimulant in the treatment of cancer. See Rosenthal, 1980, BCG Vaccine: Tuberculosis-Cancer, P.S.G. Publishing, Littleton, Mass. and Lamoureux et al., 1976, BCG in Cancer Immunotherapy, Grune & Stratton, New York.
In the preparation of these vaccines, M. bovis BCG is grown in a suitable nutrient medium and then harvested. However, during the growth phase, the cells of M. bovis BCG tend to aggregate, making the production of a homogenous vaccine very difficult. Please see Hardham and James, Microbios Letters 13: 33-42 (1980).
This paper suggests suppressing the aggregation by the inclusion of a wetting agent in the growth medium, but the suppression of aggregation, which is marginal, ceases as soon as the wetting agent is removed. It was disclosed that there is a covering material, possibly a polysaccharide, which appears to hold the cells together in aggregates. Other investigators also observed the aggregation of BCG cells. See for example Davis et al., 1968, Microbiology, Harper & Row, Evanston, IL, p.845.
The tendency of BCG cells to aggregate during the growth phase makes the standardization, evaluation and formulation of the vaccine problematic. For example, vaccine efficacy for both tuberculosis prophylaxis and cancer immunotherapy has been attributed to the presence of living cells in the vaccine, and adverse effects, including enhancement of tumor growth, have been related to the quantity of dead cells. Please see Youmans and Youmans, 1971, Immunization in Tuberculosis, DHEW Publ. No. (NIH) 72-68, pp. 219-236; Hersch et al., JAMA 235: 646-650 (1976), and Werner et al., Bull. Inst. Pasteur 75: 5-84 (1977). For these reasons, dispersal of BCG aggregates into smaller particles or single cells would therefore enable better evaluation of the vaccine at various stages of production and, most important, would increase the number of therapeutic doses per lot by increasing the number of colony-forming units (CFU) per unit volume of vaccine.